1. Technical Field
The present invention relates to agents for inhibiting adsorption of proteins on the liposome surface.
Further, the invention relates to agents for preventing liposome agglutination.
Furthermore, the invention is concerned with liposomes on which adsorption of proteins is inhibited and which are agglutination-free and a method for preparing the same.
2. Prior Art
Use of liposomes as a carrier for water-soluble or fat-soluble drugs has widely been attempted (Gregoriadis, et al., Ann. N.Y. Acad. Sci., 446, 319 (1985)). Use of liposomes as artificial erythrocytes by incorporating hemoglobin, the oxygen carrier for animals, in the inner aqueous space of liposomes has also been attempted (Japanese Patent Application Laid-Open to Public 178521/1987). Liposome membrane-constituting materials of the liposomes used in these attempts, however, were those composed only of natural or synthetic lipids such as phospholipids and cholesterol.
In order to use liposomes as a carrier for drugs it is necessary to introduce the liposomes into blood vessels in the living body. However, the liposomes composed only of lipids which were conventionally employed were encountered with problems of adsorbing plasma-constituting proteins of the living body (for example, albumin, globulin and fibrinogen) which results in mutual agglutination of the liposomes. The problems were considerable especially of the liposomes which particle size exceeds 0.1 .mu.m. Particle size of the liposomes generally employed is usually 0.1 .mu.m-1 .mu.m. That particle size will not be an obstacle in passing through the blood vessels in the living body because the capillary blood vessels have inner diameter as large as several .mu.m. However, if the liposomes are agglutinated by adsorbing plasma-constituting proteins, the size of the agglutinates becomes tens of micrometers. If the agglutination occurs in the blood vessel, agglutinates of the liposomes will plug the blood vessel to inhibit blood flow possibly causing death of the living body.
Particularly when liposomes are used as artificial erythrocytes, a large dose of liposomes should be administered so that the problem of liposome agglutination in plasma was not negligible. Heretofore, however, there has been developed no technique at all for preventing the agglutination of liposomes in plasma.
In addition, when liposomes are introduced into the living body, antibody protein (immunoglobulin) to the liposome which is an antigen will be adsorbed on the liposomes to produce foreign body recognition in the phagocytes (macrophage) with a result that the liposomes will be included in the macrophage and disappear within a short period of time. Therefore, inhibition of the protein adsorption on liposomes could also delay disappearance of the liposomes in plasma.
It is also noted that the hemoglobin concentration in natural erythrocytes is approximately 30%; as the volume ratio of erythrocytes to the whole blood (hematocrit) is approximately 50%, the hemoglobin concentration in the whole blood is approximately 15%. Accordingly, in the case of artificial erythrocytes which are formed by enclosing hemoglobin in the liposome smaller in particle size than natural erythrocytes, the volume ratio of artificial erythrocytes in an artificial erythrocyte suspension will exceed 50% when the hemoglobin concentration in the artificial erythrocyte suspension is 15%, unless an aqueous solution of hemoglobin with a hemoglobin concentration of 30% or more is subjected to liposome formation. Such suspension, which is poorly fluidized, will produce adverse effects upon circulatory dynamism when administered. In this respect, it is desirable to encapsulate a large amount of hemoglobin in the inner aqueous space of liposomes using lipid in an amount as small as possible. In other words, a method for preparing artificial erythrocytes with a high encapsulation efficiency is desirable. By the dialysis method or the reverse phase method, however, it is difficult to form liposomes of an aqueous solution of hemoglobin with a higher hemoglobin concentration (30% or more) and a higher viscosity. Also by the lamina method in which a liposome-forming lipid is uniformly dissolved in an organic solvent, then the organic solvent is removed and an aqueous solution is added to the lamina of the lipid thus formed to a dispersion, the hydration and dispersion cannot easily be accomplished by the addition of an aqueous solution because the liposome-forming lipid after removal of the organic solvent has been solidified or nearly in loss of fluidity. When the aqueous solution is an aqueous solution of hemoglobin with a high concentration, the proportion of the water combined with the globin protein is high, and the amount of the free water available for hydration of the lipid is small. Thus, liposome formation at a high efficiency was difficult. Therefore, an object of the invention is to provide agents for inhibiting adsorption of proteins on the liposome surface, agents for preventing liposome agglutination, liposomes on which adsorption of proteins in plasma is inhibited and a method for preparing the same. A further object of the invention is to provide a method for preparing artificial erythrocytes comprising forming liposomes of a highly-concentrated hemoglobin at a high efficiency.